RESUMO
Obesity is a major cause for a spectrum of metabolic syndrome-related diseases that include insulin resistance, type 2 diabetes, and steatosis of the liver. Inflammation elicited by macrophages and other immune cells contributes to the metabolic abnormalities in obesity. In addition, coagulation activation following tissue factor (TF) upregulation in adipose tissue is frequently found in obese patients and particularly associated with diabetic complications. Genetic and pharmacological evidence indicates that TF makes significant contributions to the development of the metabolic syndrome by signaling through G protein-coupled protease activated receptors (PARs). Adipocyte TF-PAR2 signaling contributes to diet-induced obesity by decreasing metabolism and energy expenditure, whereas hematopoietic TF-PAR2 signaling is a major cause for adipose tissue inflammation, hepatic steatosis and inflammation, as well as insulin resistance. In the liver of mice on a high fat diet, PAR2 signaling increases transcripts of key regulators of gluconeogenesis, lipogenesis and inflammatory cytokines. Increased markers of hepatic gluconeogenesis correlate with decreased activation of AMP-activated protein kinase (AMPK), a known regulator of these pathways and a target for PAR2 signaling. Clinical markers of a TF-induced prothrombotic state may thus indicate a risk in obese patient for developing complications of the metabolic syndrome.
Assuntos
Inflamação/imunologia , Síndrome Metabólica/imunologia , Obesidade/imunologia , Receptor PAR-2/imunologia , Transdução de Sinais/imunologia , Tromboplastina/imunologia , Animais , Humanos , Fatores Imunológicos/imunologia , Modelos ImunológicosRESUMO
INTRODUCTION: The first descriptions of transient global amnesia (TGA) were made in 1956 and 1958. Considering the large number of TGA reported since these original descriptions, it is not conceivable that TGA arose as a new entity in the mid-20th century. Many authors thus tried to understand why it had not been described by the authors of the late 19th and early 20th century. It was considered that TGA "was immersed in the literature on psychogenic amnesia" (Hodges, 1991) and particularly hysterical amnesia. But, can we consider that confusion between transient global amnesia and psychogenic amnesia truly existed? METHODS: The book of Paul Sollier, a student of Charcot and Ball, emphasizes the memory problems that were discussed in the second part of the 19th century. RESULTS: The author presents a clear differentiation between hysterical amnesia and amnesia triggered by an emotional shock. The cases he proposed include characteristic descriptions of transient global amnesia observed after a violent emotional shock. Sollier, like Ball and his student Rouillard, also considered transient amnesias such as post-traumatic amnesias occurring after mild head trauma. The triggering role was assigned to the "moral emotion" that can provoke a modification of the encephalic circulation. DISCUSSION: While TGA was not yet recognized as an entity, some French neurologists of the 19th century reported cases of temporary amnesias different from hysterical amnesia and occurring after an emotional shock, which were the first observations of the entity later recognized as TGA.
Assuntos
Amnésia Global Transitória/história , Amnésia/história , Neurologia/história , Amnésia/classificação , Amnésia/diagnóstico , Amnésia/etiologia , Amnésia/psicologia , Amnésia Global Transitória/classificação , Amnésia Global Transitória/diagnóstico , Amnésia Global Transitória/etiologia , Lesões Encefálicas/complicações , Lesões Encefálicas/psicologia , Emoções , França , História do Século XIX , História do Século XX , Humanos , Modelos Neurológicos , Modelos Psicológicos , Transtornos de Estresse Pós-Traumáticos/complicações , Transtornos de Estresse Pós-Traumáticos/psicologiaRESUMO
Altered expression of proteins of the fibrinolytic and coagulation cascades in obesity may contribute to the cardiovascular risk associated with this condition. In spite of this, the zymogenic nature of some of the molecules and the presence of variable amounts of activators, inhibitors, and cofactors that alter their activity have made it difficult to accurately monitor changes in the activities of these proteins in tissues where they are synthesized. Thus, as a first approach to determine whether tissue factor (TF) expression is altered in obesity, this study examined changes in TF mRNA in various tissues from lean and obese (ob/ob and db/db) mice. TF gene expression was elevated in the brain, lung, kidney, heart, liver, and adipose tissues of both ob/ob and db/db mice compared with their lean counterparts. In situ hybridization analysis indicated that TF mRNA was elevated in bronchial epithelial cells in the lung, in myocytes in the heart, and in adventitial cells lining the arteries including the aortic wall. Obesity is associated with insulin resistance and hyperinsulinemia, and administration of insulin to lean mice induced TF mRNA in the kidney, brain, lung, and adipose tissue. These observations suggest that the hyperinsulinemia associated with insulin-resistant states, such as obesity and noninsulin-dependent diabetes mellitus, may induce local TF gene expression in multiple tissues. The elevated TF may contribute to the increased risk of atherothrombotic disease that accompanies these conditions.
Assuntos
Regulação da Expressão Gênica , Obesidade/genética , Tromboplastina/genética , Tecido Adiposo/química , Animais , Química Encefálica , Regulação da Expressão Gênica/efeitos dos fármacos , Hiperinsulinismo/complicações , Hibridização In Situ , Insulina/farmacologia , Resistência à Insulina , Rim/química , Fígado/química , Pulmão/química , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Obesos , Miocárdio/química , Obesidade/complicações , RNA Mensageiro/análise , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
BACKGROUND: Although the association between insulin resistance and cardiovascular risk is well established, the underlying molecular mechanisms are poorly understood. The antifibrinolytic molecule plasminogen activator inhibitor 1 (PAI-1) is a cardiovascular risk factor that is consistently elevated in insulin-resistant states such as obesity and non-insulin-dependent diabetes mellitus (NIDDM). The strong positive correlation between this elevated PAI-1 and the degree of hyperinsulinemia not only implicates insulin itself in this increase, but also suggests that PAI-1 is regulated by a pathway that does not become insulin resistant. The data in this report supports this hypothesis. MATERIALS AND METHODS: We show that insulin stimulates PAI-1 gene expression in metabolically insulin-resistant ob/ob mice and in insulin-resistant 3T3-L1 adipocytes. Moreover, we provide evidence that glucose transport and PAI-1 gene expression are mediated by different insulin signaling pathways. These observations suggest that the compensatory hyperinsulinemia that is frequently associated with insulin-resistant states, directly contribute to the elevated PAI-1. CONCLUSIONS: These results provide a potential mechanism for the abnormal increases in cardiovascular risk genes in obesity, NIDDM, and polycystic ovary disease.
Assuntos
Adipócitos/efeitos dos fármacos , Resistência à Insulina , Insulina/farmacologia , Inibidor 1 de Ativador de Plasminogênio/genética , Regulação para Cima/efeitos dos fármacos , Adipócitos/metabolismo , Animais , Transporte Biológico/efeitos dos fármacos , Glicemia/metabolismo , Doenças Cardiovasculares/complicações , Doenças Cardiovasculares/genética , Doenças Cardiovasculares/metabolismo , Linhagem Celular , Desoxiglucose/metabolismo , Diabetes Mellitus Tipo 2/complicações , Diabetes Mellitus Tipo 2/genética , Diabetes Mellitus Tipo 2/metabolismo , Hiperinsulinismo/complicações , Hiperinsulinismo/genética , Hiperinsulinismo/metabolismo , Hibridização In Situ , Insulina/sangue , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Obesos , Obesidade/complicações , Obesidade/genética , Obesidade/metabolismo , Inibidor 1 de Ativador de Plasminogênio/sangue , Inibidor 1 de Ativador de Plasminogênio/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Transdução de Sinais/efeitos dos fármacosRESUMO
In this chapter, we summarize our studies on plasminogen activator inhibitor 1 (PAI-1), tissue factor, and transforming growth factor beta (TGF-beta) expression in obesity, using genetically obese mice as a model. These studies emphasize the key role played by the adipocyte, a cell whose numbers, size, and metabolic activity are grossly altered in obesity/NIDDM. They also implicate multiple cytokines, hormones, and growth factors in the abnormal expression of these and perhaps other hemostatic genes by adipocytes in obesity/NIDDM. These studies demonstrate that tumor necrosis factor alpha (TNF-alpha) plays a central role in the expression of hemostatic genes in this disorder.
Assuntos
Diabetes Mellitus Tipo 2/genética , Diabetes Mellitus/genética , Hemostasia/genética , Camundongos Obesos/genética , Obesidade/genética , Adipócitos/fisiologia , Animais , Regulação da Expressão Gênica , Humanos , Camundongos , Inibidor 1 de Ativador de Plasminogênio/genética , Fator de Crescimento Transformador beta/genética , Fator de Necrose Tumoral alfa/fisiologiaAssuntos
Hemostasia/genética , Obesidade/complicações , Obesidade/genética , Doenças Vasculares/etiologia , Doenças Vasculares/genética , Tecido Adiposo , Animais , Diabetes Mellitus Tipo 2 , Modelos Animais de Doenças , Expressão Gênica , Humanos , Camundongos , Inibidor 1 de Ativador de Plasminogênio , Fator de Crescimento Transformador beta , Fator de Necrose Tumoral alfaRESUMO
Obesity is associated with a cluster of abnormalities, including hypertension, insulin resistance, hyperinsulinemia, and elevated levels of both plasminogen activator inhibitor 1 (PAI-1) and transforming growth factor beta (TGF-beta). Although these changes may increase the risk for accelerated atherosclerosis and fatal myocardial infarction, the underlying molecular mechanisms remain to be defined. Although tumor necrosis factor alpha (TNF-alpha) has been implicated in the insulin resistance associated with obesity, its role in other disorders of obesity is largely unknown. In this report, we show that in obese (ob/ob) mice, neutralization of TNF-alpha or deletion of both TNF receptors (TNFRs) results in significantly reduced levels of plasma PAI-1 antigen, plasma insulin, and adipose tissue PAI-1 and TGF-beta mRNAs. Studies in which exogenous TNF-alpha was infused into lean mice lacking individual TNFRs indicate that TNF-alpha signaling of PAI-1 in adipose tissue can be mediated by either the p55 or the p75 TNFR. However, TNF-alpha signaling of TGF-beta mRNA expression in adipose tissue is mediated exclusively via the p55 TNFR. Our results suggest that TNF-alpha is a common link between the insulin resistance and elevated PAI-1 and TGF-beta in obesity. The chronic elevation of TNF-alpha in obesity thus may directly promote the development of the complex cardiovascular risk profile associated with this condition.
Assuntos
Obesidade/sangue , Inibidor 1 de Ativador de Plasminogênio/metabolismo , Fator de Necrose Tumoral alfa/metabolismo , Animais , Regulação da Expressão Gênica , Resistência à Insulina , Camundongos , Camundongos Obesos , Obesidade/genética , Inibidor 1 de Ativador de Plasminogênio/genética , Receptores do Fator de Necrose Tumoral/metabolismo , Fatores de Risco , Fator de Crescimento Transformador beta/metabolismo , Fator de Necrose Tumoral alfa/genéticaRESUMO
Altered expression of proteins of the fibrinolytic and coagulation cascades in obesity may contribute to the cardiovascular risk associated with this condition. We previously reported that plasminogen activator inhibitor 1 (PAI-1) is dramatically up-regulated in the plasma and adipose tissues of genetically obese mice. This change may disturb normal hemostatic balance and create a severe hypofibrinolytic state. Here we show that tissue factor (TF) gene expression also is significantly elevated in the epididymal and subcutaneous fat pads from ob/ob mice compared with their lean counterparts, and that its level of expression in obese mice increases with age and the degree of obesity. Cell fractionation and in situ hybridization analysis of adipose tissues indicate that TF mRNA is increased in adipocytes and in unidentified stromal vascular cells. Transforming growth factor beta (TGF-beta) is known to be elevated in the adipose tissue of obese mice, and administration of TGF-beta increased TF mRNA expression in adipocytes in vivo and in vitro. These observations raise the possibility that TF and TGF-beta may contribute to the increased cardiovascular disease that accompanies obesity and related non-insulin-dependent diabetes mellitus, and that the adipocyte plays a key role in this process. The recent demonstration that TF also influences angiogenesis, cell adhesion, and signaling suggests that its exact role in adipose tissue physiology/pathology, may be complex.
Assuntos
Tecido Adiposo/metabolismo , Expressão Gênica , Obesidade/genética , Tromboplastina/genética , Adipócitos/metabolismo , Animais , Fibrinólise , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Obesos , Obesidade/metabolismo , Inibidor 1 de Ativador de Plasminogênio/biossíntese , RNA Mensageiro/metabolismo , Trombose/genética , Fator de Crescimento Transformador beta/fisiologiaAssuntos
Adipócitos/metabolismo , Obesidade/metabolismo , Inibidor 1 de Ativador de Plasminogênio/fisiologia , Inibidores de Serina Proteinase/fisiologia , Animais , Doenças Cardiovasculares/metabolismo , Ácidos Graxos não Esterificados/fisiologia , Regulação da Expressão Gênica , Humanos , Insulina/fisiologia , Inibidor 1 de Ativador de Plasminogênio/metabolismo , RNA Mensageiro/análise , Tromboplastina/metabolismo , Tromboplastina/fisiologia , Trombose/metabolismo , Fator de Crescimento Transformador beta/metabolismo , Fator de Crescimento Transformador beta/fisiologia , Triglicerídeos/fisiologia , Fator de Necrose Tumoral alfa/fisiologiaRESUMO
Plasminogen activator inhibitor-1 is elevated in obesity and may be a risk factor for obesity/NIDDM related cardiovascular disease. In spite of this, little is known about the tissue and cellular origin of elevated PAI-1 in obesity or of the mediators and molecular mechanisms that regulate it. We have begun to systematically address these issues using genetically obese (ob/ob, db/db) mice. Plasma PAI-1 levels were 5-fold higher in obese mice compared to their lean counterparts. Subsequent RT-PCR and in situ hybridization studies suggest that the increased plasma PAI-1 originates primarily from the adipocyte in response to chronically elevated levels of tumor necrosis factor-alpha (TNF-alpha), insulin, and transforming growth factor-beta (TGF-beta). Thus, the signals and mechanisms that lead to elevated plasma PAI-1 observed in obesity are complex, and appear to involve interactions between multiple mediators and the adipose tissue itself.
Assuntos
Diabetes Mellitus Tipo 2/sangue , Diabetes Mellitus/sangue , Obesidade , Inibidor 1 de Ativador de Plasminogênio/sangue , Animais , Diabetes Mellitus/genética , Diabetes Mellitus Tipo 2/genética , Humanos , Camundongos , Camundongos Obesos , Modelos Genéticos , Fatores de Risco , Fator de Crescimento Transformador beta/sangue , Fator de Necrose Tumoral alfa/metabolismoRESUMO
The disturbances in the balance of pro- and antifibrinolytic activity, as observed in AAA and obesity, respectively, have considerable potential for influencing both intra- and extravascular fibrinolytic events and may be causally related to the development of vascular disease. For example, the wall of the aortic atherosclerotic aneurysm seems to host an uneven distribution and imbalanced expression of the various components of the fibrinolytic system. The sites of increased proteolytic activity may contribute to localized neovascularization and promote the rapid breakdown of ECM components, which result in mural weakening and eventual rupture of untreated aortic aneurysms. On the other hand, the disturbance of the normal hemostatic balance observed in obesity appears to result from the elevated expression of PAI-1 by the adipose tissue. Our data strongly suggest that the adipocyte is one of the primary cells in the adipose tissue capable of expressing PAI-1 both in obesity, and in response to cytokines and hormones like TNF-alpha and insulin. Since both TNF-alpha and insulin are known to increase in obesity, the elevated levels of PAI-1 observed in the plasma of obese individuals may result from TNF-alpha and/or insulin induction of PAI-1 in the adipose tissue itself.
Assuntos
Arteriosclerose/genética , Fibrinólise/genética , Obesidade/genética , Inibidor 1 de Ativador de Plasminogênio/genética , Aneurisma , Animais , Arteriosclerose/sangue , Regulação da Expressão Gênica , Humanos , Camundongos , Camundongos Obesos , Obesidade/sangueRESUMO
BACKGROUND: Tumor necrosis factor-alpha (TNF-alpha) is chronically elevated in the adipose tissue from obese humans and mice. This increase in TNF-alpha contributes to the insulin resistance, elevated plasminogen activator inhibitor-1 (PAI-1) levels, and cardiovascular complications associated with obesity and noninsulin-dependent diabetes (NIDDM). PAI-1 gene expression in adipose tissue is also stimulated by transforming growth factor-beta (TGF-beta). Experiments were performed to determine whether TGF-beta is regulated by TNF-alpha and elevated in obesity. MATERIALS AND METHODS: The concentration of TGF-beta and PAI-1 mRNA in murine adipose tissue and cultured 3T3-L1 adipocytes was determined by quantitative reverse transcription polymerase chain reaction (RT-PCR), and the cellular localization of these molecules was evaluated using in situ hybridization and cell fractionation. Total TGF-beta protein was determined by employing an ELISA assay. RESULTS: TGF-beta mRNA and protein were increased in the adipose tissue from two different strains of genetically obese mice (i.e., ob/ob and db/db), compared with their lean counterparts. This increase in TGF-beta may result from TNF-alpha since TNF-alpha increased TGF-beta mRNA expression in the adipose tissue of lean mice and stimulated TGF-beta production by cultured adipocytes. Administration of TGF-beta increased PAI-1 antigen in the plasma and PAI-1 mRNA in the adipocytes of lean mice, and enhanced the rate of PAI-1 synthesis by adipocytes in vitro. CONCLUSIONS: TNF-alpha contributes to the elevated TGF-beta expression demonstrated in the adipose tissue of obese mice. A potential role for TGF-beta in the increased PAI-1 and vascular pathologies associated with obesity/NIDDM is suggested.
Assuntos
Tecido Adiposo/química , Obesidade/metabolismo , Fator de Crescimento Transformador beta/análise , Células 3T3 , Animais , Fracionamento Celular , Regulação da Expressão Gênica/fisiologia , Insulina/farmacologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Obesos , Obesidade/genética , Inibidor 1 de Ativador de Plasminogênio/análise , Inibidor 1 de Ativador de Plasminogênio/sangue , Inibidor 1 de Ativador de Plasminogênio/genética , RNA Mensageiro/análise , Fator de Crescimento Transformador alfa/farmacologia , Fator de Crescimento Transformador beta/genéticaRESUMO
BACKGROUND: Although elevated plasminogen activator inhibitor-1 (PAI-1) is associated with obesity and may be a risk factor for cardiovascular disease, the mechanism(s) that lead to this elevation, and the tissue/cellular origins of this increase, remain to be defined. In this report, we have addressed these questions using genetically obese mice (ob/ob) and their lean counterparts (+/?). MATERIALS AND METHODS: PAI-1 activity and antigen levels were determined using a tissue-type plasminogen activator (t-PA) binding assay and Western blotting. The concentration of PAI-1 mRNA in tissues was determined by quantitative reverse transcriptase-polymerase chain reaction (RT-PCR), and the cellular localization of PAI-1 was evaluated using in situ hybridization, immunohistochemistry, and cell fractionation. RESULTS: PAI-1 activity was approximately 4-fold higher in plasma from ob/ob mice than in that obtained from their lean counterparts, and this difference increased further with age (i.e., 6-fold at 3 months). PAI-1 mRNA levels were elevated 4- to 5-fold in the adipose tissues of obese mice, and these differences in mRNA also increased with age. The elevated PAI-1 mRNA in the adipose tissues of obese mice was localized to mature adipocytes as well as to vascular smooth muscle cells and occasional endothelial cells. Obesity is often associated with hyperinsulinemia, and acute injection of insulin into lean mice increased PAI-1 mRNA 6- to 8-fold in the epididymal fat in cells that morphologically resembled adipocytes. Insulin did not increase PAI-1 in large vessel endothelial or smooth muscle cells. The adipocyte response to insulin was confirmed in cell culture studies where PAI-1 synthesis by mature 3T3-L1 adipocytes was increased 5- to 6-fold by insulin. CONCLUSIONS: Our results suggest that elevated PAI-1 associated with obesity may result in part from insulin-induced induction of PAI-1 specifically by adipocytes within the fat itself.
Assuntos
Obesidade/metabolismo , Inibidor 1 de Ativador de Plasminogênio/metabolismo , Células 3T3 , Tecido Adiposo/metabolismo , Animais , Expressão Gênica/efeitos dos fármacos , Insulina/metabolismo , Insulina/farmacologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Inibidor 1 de Ativador de Plasminogênio/sangue , Inibidor 1 de Ativador de Plasminogênio/genética , RNA Mensageiro/metabolismoRESUMO
Although elevated plasma plasminogen activator inhibitor 1 (PAI-1) is associated with obesity, very little is known about its tissue or cellular origin, or about the events that lead to increased PAI-1 levels under obese conditions. Since TNF-alpha is increased in rodents both during obesity and in response to endotoxin treatment, we examined the effects of these agents on PAI-1 gene expression in the adipose tissue of CB6 mice. In untreated mice, PAI-1 mRNA was detected in both mature adipocytes and in stromal vascular cells. Both TNF-alpha and endotoxin significantly increased PAI-1 mRNA in the adipose tissue, peaking at 3-8 h. In situ hybridization analysis of adipose tissue from untreated mice revealed a weak signal for PAI-1 mRNA only in the smooth muscle cells within the vascular wall. In contrast, after endotoxin or TNF-alpha treatment, PAI-1 mRNA also was detected in adipocytes and in adventitial cells of vessels. Endotoxin also induced PAI-1 in endothelial cells, while TNF-alpha additionally induced it in smooth muscle cells. Mature 3T3-L1 adipocytes in culture also expressed PAI-1 mRNA, and its rate of synthesis was also upregulated by TNF-alpha. These studies suggest that the adipose tissue itself may be an important contributor to the elevated PAI-1 levels observed in the plasma under obese conditions.
Assuntos
Tecido Adiposo/metabolismo , Regulação da Expressão Gênica/efeitos dos fármacos , Lipopolissacarídeos/farmacologia , Inibidor 1 de Ativador de Plasminogênio/genética , Fator de Necrose Tumoral alfa/farmacologia , Células 3T3 , Adipócitos/química , Animais , Sequência de Bases , Células Cultivadas , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Dados de Sequência Molecular , Músculo Liso Vascular/química , Músculo Liso Vascular/citologia , Inibidor 1 de Ativador de Plasminogênio/análise , RNA Mensageiro/análiseRESUMO
We report the sequence of 8.1 kb of DNA containing the 3' end of one and seven other complete intronless globin genes from the YWVZ/7B locus of the dipteran Chironomus thummi thummi. One of these (ctt-v) appears to be a pseudogene by virtue of a premature termination codon, whereas the others encode apparently functional globin polypeptides. taken together with previously published data, the C. th. thummi YWVZ/7B locus codes for at least 11 globins, five of which differ from one another by no more than two amino acids. In contrast only nine globin genes are found in a comparable genomic clone isolated from C. th. piger. As indicated by sequence alignment, this difference in copy number can be attributed to a loss of one gene (fusion of globin genes 7B8 and 7B10) in the piger lines, coupled with a gain (globin gene 7B9) in the thummi lineage. Comparisons between the thummi and piger sequences showed that YWVZ/7B intergenic regions have maintained a level of 91% similarity since the thummi/piger divergence: most differences are simply due to single base substitutions or insertion/deletion events in either the thummi or the piger DNA, but three instances of partially overlapping deletions were also detected. A phylogenetic analysis of YWVZ/7B gene products was conducted, from which a plausible reconstruction of the evolutionary history of the locus was obtained. In addition, alignment of globin 7B amino acid sequences suggested that globin genes 7B2 and 7B3 (reported at the protein and cDNA level, respectively, but not contained on the C. th. thummi or C. th. piger genomic clones) are possibly chimeric genes. Given the trend toward expansion of the C. thummi globin gene family in general and of the globin 7B subfamily in particular, we propose that increased copy number of these genes has been positively selected as a mechanism to achieve a high Hb concentration in the larval hemolymph.
Assuntos
Chironomidae/genética , Genes de Insetos , Globinas/genética , Família Multigênica , Filogenia , Sequência de Aminoácidos , Animais , Sequência de Bases , Chironomidae/classificação , Dados de Sequência Molecular , Alinhamento de Sequência , Homologia de Sequência , Especificidade da EspécieRESUMO
We determined that exposure of cultured lung fibroblasts (HEL-299) to recombinant human interleukin-6 (0-400 ng/ml) resulted in a dose- and time-dependent increase in secreted and cell lysate PAI-1 and total tPA levels (maximal increase of 2.6-fold and 1.7-fold, respectively). Specificity of this response was indicated when increases in PAI-1 levels were inhibited by neutralizing polyclonal antibodies to IL-6, but not with non-specific antibodies. Inhibition of the response to IL-6 by cycloheximide and alpha-amanitin indicates that increases in PAI-1 are dependent on both protein and RNA synthesis. The addition of IL-6 to HEL-299 cells also stimulated a dose- and time-dependent increase in steady-state PAI-1 mRNA levels (3.8 to 15.1 pg/micrograms total RNA by 24 h). A rapid increase (5-6-fold) in PAI-1 mRNA levels was found between 3 and 12 h. Nuclear run-on assays using a maximum dose of IL-6 showed that IL-6 increases a 4-fold rate of transcription of the PAI-1 gene. We further showed that LPS induces a 70% increase in secreted IL-6 and a 50% increase in PAI-1 protein levels. Increasing doses of anti-IL-6 completely blocked the effect of LPS on PAI-1 while non-specific antibodies had no effect. These studies suggest an autocrine role for IL-6 in regulating localized proteolysis and modulating tissue remodeling during acute inflammatory conditions by fibroblasts.
Assuntos
Interleucina-6/farmacologia , Inibidor 1 de Ativador de Plasminogênio/genética , Ativadores de Plasminogênio/genética , Plasminogênio/metabolismo , Células Cultivadas , Relação Dose-Resposta a Droga , Fibroblastos/efeitos dos fármacos , Fibroblastos/metabolismo , Regulação da Expressão Gênica , Glucocorticoides/farmacologia , Humanos , Inibidor 1 de Ativador de Plasminogênio/biossíntese , Ativadores de Plasminogênio/biossíntese , RNA Mensageiro/análise , Proteínas Recombinantes/farmacologiaRESUMO
Interleukin-6 (IL6) is produced by different cell types, including monocytes and fibroblasts. We show that recombinant human IL6 (rhIL6) and chick fibroblast conditioned medium stimulate plasma fibronectin (PFn) and PFn mRNA production by cultured chick hepatocytes in a dose-dependent manner. Lipopolysaccharide (LPS) treatment of fibroblast cultures induces higher levels of the PFn stimulating activity. These effects are blocked by preincubation of either rhIL6 or LPS-stimulated chick fibroblast conditioned medium with anti-rhIL6 antibody before treatment of hepatocytes, indicating that the conditioned medium contains chick fibroblast-derived IL-6 (cfIL6). Further, LPS induces fibroblast production of a proportional increase in cfIL6 detectable by a human IL6 ELISA. cfIL6 maximally stimulates chick hepatocyte PFn production by 24 h (4.5-fold). Dexamethasone acts more rapidly, but maximal stimulation is only 2.3-fold. Hepatocyte Fn mRNA levels are even more substantially stimulated by dexamethasone and cfIL6 (up to 8.9- and 18.5-fold by 12 h, declining to 2.3 and 4.2-fold by 24 h, respectively). The effect cfIL6 with or without dexamethasone on hepatocyte PFn levels are comparable. These observations are consistent with the role of IL6 as a major mediator of acute phase protein production.
Assuntos
Proteínas de Fase Aguda/biossíntese , Fibroblastos/metabolismo , Fibronectinas/biossíntese , Interleucina-6/metabolismo , Fígado/metabolismo , Animais , Células Cultivadas/efeitos dos fármacos , Galinhas , Meios de Cultivo Condicionados , Dexametasona/farmacologia , Fibronectinas/genética , RNA Mensageiro/análise , Proteínas Recombinantes/metabolismoRESUMO
We isolated and sequenced a 432 bp cDNA to cAT-III, that encoded 115 nucleotides of 5' untranslated sequence, a 17 amino acid long signal peptide and residues 1-88 of the mature protein, and used it to prepare a probe for measuring and correlating the developmental changes of steady-state cAT-III mRNA levels with known changes in antigen levels. Densitometric analysis of nuclease protection (n = 2), Northern blot (n = 4), and slot blots (n = 3) of total RNA from chick livers of 16-day-old embryos to 6-day-old chicks showed a 2.6 +/- 0.5-fold increase in steady-state cAT-III mRNA levels. Assay of functional mRNA levels by in vitro translation of poly(A)+ RNA and specific immunoprecipitation of 35S-Met-labelled cAT-III was comparable to RNA analysis (16-day-old embryos vs. 10-day-old hatchlings). We evaluated whether there were developmental differences in post-translational secretion which may also contribute to the regulation of the circulating level of this protein. Pulse-chase studies of freshly-isolated hepatocytes from 16-day-old embryos and 10-day-old hatchlings maintained in suspension demonstrated a approx. 5.0-5.5-fold increase in cAT-III levels at steady-state secretion. The above findings indicate that changes in circulating cAT-III levels during late embryonic development are primarily due to increased abundance of cAT-III mRNA. In addition, we postulate that post-translational intracellular processing may account for further differences in circulating protein levels.